Automation protocol for high-efficiency and high-quality genomic DNA extraction from Saccharomyces cerevisiae

Although many protocols have been previously developed for genomic DNA (gDNA) extraction from S. cerevisiae, to take advantage of recent advances in laboratory automation and DNA-barcode sequencing, there is a need for automated methods that can provide high-quality gDNA at high efficiency. Here, we describe and demonstrate a fully automated protocol that includes five basic steps: cell wall and RNA digestion, cell lysis, DNA binding to magnetic beads, washing with ethanol, and elution. Our protocol avoids the use of hazardous reagents (e.g., phenol, chloroform), glass beads for mechanical cell disruption, or incubation of samples at 100°C (i.e., boiling). We show that our protocol can extract gDNA with high efficiency both from cells grown in liquid culture and from colonies grown on agar plates. We also show results from gel electrophoresis that demonstrate that the resulting gDNA is of high quality.


Preparation of 4x Lysate Buffer Preparation of Magnetic Beads Stock Preparation of Magnetic Bead/PEG-NaCl Binding Buffer
Fill Conical Tube to the 40 mL mark with Nuclease-Free Water, then vortex to mix every 1-2 minutes until PEG goes into solution.
Vortex Magnetic Beads Stock (from previous section) to mix thoroughly.
Add Magnetic Bead Stock:

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For extraction of gDNA from liquid culture, add 3 mL Magnetic Beads Stock to the 50 mL Conical Tube containing the PEG/NaCl solution.

29.2
For extraction of gDNA from yeast colonies, add 1 mL Magnetic Beads Stock to the 50 mL Conical Tube containing the PEG/NaCl solution.
Fill Conical Tube to the 50 mL mark with Nuclease-Free Water, and gently mix until solution is a uniform brown color.
Wrap in foil (or place in dark container) and store at 4 °C, mix well by vortexing before use.
Centrifuge at 3700 g for 10 min at 10 °C.
Remove supernatant from each well using automated liquid handler.

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Use modified labware definition or other constraints on automated pipetting to set a minimum distance of 2.5 mm between the bottom of each well and the tip of each pipette.

34.2
The resulting volume of the remaining cell pellet and residual liquid should be approximately 50 µL.
Seal Spin Plate using Aluminum Foil Lid and Rubber Roller.After removing most of the supernatant with large-volume tips, use smaller-volume tips (e.g., 50 µL) to remove any residual supernatant from the bottom of each well.
Allow magnetic bead pellets to dry for 5 minutes at room temperature.
Move the Reaction Plate from magnet base to an automated thermal-regulated microplate shaker with temperature set to 60 °C.
Add 100 µL Nuclease-Free Water to each well (note: can also use EB) Resuspend beads by repeated aspiration and dispense (12 times).
Incubate at 60 °C for 10 s while shaking at 1000 rpm.
Incubate at 60 °C for 7 minutes while shaking at 400 rpm.
Move the Reaction Plate back to the magnet base.
Wait for 5 minutes.
Transfer the supernatant from each well to a new, clean well in the same plate (still on the magnet base).
Wait for 5 minutes.
Transfer the supernatant with extracted DNA from each well to a well in the Elution plate.
Cover the Elution plate containing the extracted DNA samples with a Universal Lid.
no. 352098) • Microcentrifuge Tubes, 1.7 mL, RNase/DNase free (Costar, cat.no.3207/3620) Equipment: Equipment: • Automated liquid handler (Hamilton Robotics, NGS-STAR).• Centrifuge for 96-well plates.Prepare stock of Zymolyase 20T by dissolving to a concentration of 1 U/µL in 1x PBS.Divide Zymolyase solution into 800 µL aliquots and store at -20 °C until use.Prepare 0.001 % stock of 2-Mercaptoethanol by diluting 1000-fold into 1x PBS.Store at 4 °C until use.Add 22.72 mL 1x PBS Add one aliquot (800 µL) of Zymolyase Stock, 1 U/µL Add 160 µL 0.001 % 2-Mercaptoethanol Add 320 µL RNase A/T1 mix Prepare fresh before each use Add 30 mL Nuclease-Free Water.Add 60 mL 20% SDS Add 4 mL 0.5 mol/L EDTA.Add 6 mL 1 mol/L TRIS-HCL, pH 8.0 Store at 4 °C until use.Vortex Speed-Bead Magnetic beads to resuspend Divide Speed-Bead Magnetic beads into 1 mL aliquots, each in in a 1.7 mL Microcentrifuge Tube.Place micro-centrifuge tubes containing magnetic beads solutions on a 12-Tube Magnetic Separation Rack until beads are drawn to the magnet and solutions are clear.Remove supernatant.Add 1 mL 1x TE Buffer to each micro-centrifuge tube containing beads, remove from magnetic stand, and mix by vortex.Repeated steps 17-19 two additional times.Store bead stock in the dark at 4 °C.Bring bead stock to room temperature and mix well by vortexing before use.Add 9 g PEG-8000 to a new 50 mL Conical Tube.Add 10 mL 5 mol/L Sodium Chloride.Add 500 µL 1 mol/L Tris-HCl, pH8.Add 500 µL 0.5 mol/L EDTA.
Store at -20 °C.Defrost at 4 °C for 30-60 minutes before continuing with extraction.Pick up colonies from agar plate and resuspend each in 50 μL of PBS in 1-3 columns of 96-well Spin Plate.Proceed with DNA extraction from resuspended colonies as described below.Pipette 315 µL of Digestion Master Mix into each well containing a cell pellet or resuspended Preparation of yeast liquid culture for extraction Preparation of yeast colonies for extraction Yeast genomic DNA extraction.The following steps are imple… 58.1